Liposomes are vesicles produced from bilayer forming lipids and are currently of interest as drug delivery vehicles. Liposomal formulations are capable of altering the biodistribution of drugs which, in certain cases, results in reduced toxicity and/or an improvement in the therapeutic index of the drug. The biodistribution of liposomes may be altered by changing the size or lipid composition. An alternative procedure of current interest for altering biodistribution involves the conjugation of targeting vectors to the liposomal surface (see, Heath, et al., Chem. Phys. Lipids 40:347 (1986)). Such vectors should allow tissue specific accumulation of the drug. The targeting vectors usually used are antibodies specific for an antigen expressed mostly by the targeted tissue.
A number of different protocols for the conjugation of antibodies to the surface of liposomes have been developed. In one type of protocol, antibodies have been bound to liposomes which have protein A or protein G conjugated to their surfaces. See, Leserman, et al., Nature 288:602-604 (1980). Protein A and protein G bind to the Fc portion of antibodies, allowing, in principle, both antigen binding sites to be available. A disadvantage of the procedure is the difficulty in selectively derivatizing the protein A or G so that it is correctly orientated and capable of binding the antibody. Additionally, the conjugated protein A or G may, in some cases, induce an immunogenic response. Moreover, the protocols used to conjugate the protein A or G may also cause formation of ternary liposome-protein-liposome complexes.
A similar type of protocol uses avidin or streptavidin conjugated to liposomes to bind biotinylated antibodies. See, Loughrey, et al., Biochimn. Biophys. Acta 901:157-160 (1987). However, it is difficult to control aggregation with these systems since crosslinking can occur during the avidin conjugation step or on exposure to the biotinylated antibody.
A third type of protocol involves direct coupling of the antibody to the liposomal surface. Usually this is achieved by modification of the antibody's amino functions with a crosslinking agent such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP). See, Heath, et al., Proc. Natl. Acad. Sci. USA 80:1377-1381 (1983). However, such modifications are not selective and (i) may damage the antigen binding site (ii) may not orientate the antibody in such a manner that the antigen binding sites are available for binding. In addition, such liposomes may aggregate during derivitization and be rapidly cleared in vivo due to Fc receptor mediated recognition by the immune system.
Alteratively, antibodies may be coupled to the liposomal surface after mild oxidation of the carbohydrate functions located in the C.sub.H .sup.2 domain of the Fc fragment. These procedures allow selective modification of the antibody such that (i) the antigen binding sites are not damaged (ii) the antigen binding sites are always directed outward from the liposome surface (see, Domen, et al., J. Chromatography 510:293 (1990)). The most common procedure of this sort involves coupling the oxidized antibody to a liposome surface containing amino functions by reductive amination using sodium cyanoborohydride. Aggregation may result from such systems due to reductive amination occurring with the amino functions of a second antibody. A potentially more useful procedure involves conjugation of the oxidized antibody directly to hydrazide functions on the liposome surface (see, Chua, et al., Biochim. Biophys. Acta 800:291 (1984)). The disadvantage of the latter systems lies with the poor anchoring ability of short single acyl chains. In addition, the hydrazide-aldehyde reaction proceeds at a lower rate than the more commonly used thiol-maleimide reaction. Consequently, long reaction times may be required to effect adequate conjugation between the protein and liposome surfaces. Related compounds reported by Zalipsky, Bioconjugate Chem. 4:296-299 (1993), the disclosure of which are incorporated herein by reference, could potentially be used in a similar manner.
What is needed in the art is a new procedure for linking glycosylated proteins to liposomes which avoids the above problems and provides compositions capable of delivering therapeutic agents to their sites of action. Surprisingly, the present invention provides such compositions and methods.